The long-term goal of this research is to reduce the susceptibility of vulnerable cells to the deleterious effects of chronic oxidative stress. Oxidative stress is widely accepted as a primary contributor to a range of pathological conditions including certain cancers, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's), diabetic retinopathy, and age-related macular degeneration. The ubiquitin (Ub) proteolytic system (UPS) defends cells against oxidative stress by degrading damaged proteins and by regulating cytoprotective proteins. Paramount among these protective proteins is Nrf2, the master anti-oxidant transcription factor. Oxidative stress activates Nrf2 to induce the expression of anti-oxidant enzymes and factors that restore redox homeostasis. Yet, open questions remain as to the mechanism(s) by which Nrf2 is sequestered on the promoters of its target genes until redox homeostasis is restored. The studies of this application address this critical issue and offer fresh insights into how the UPS contributes to the endogenous antioxidant defense system. The foundation for this work is our recent discovery that the E2 ubiquitin conjugating enzyme, UbcM2, is a novel component of the endogenous oxidative stress response pathway. These findings have led to our overarching hypothesis that UbcM2 is in fact a multi-functional E2. It functions as a redox sensor to enhance the cytoprotective activity of Nrf2 and it plays critical roles in protein degradation and E3 ligase regulation. Three specific aims will be pursued to define the diverse functions of UbcM2. Sp Aim#1: Test the hypothesis that UbcM2 is a redox sensor protein that promotes the cytoprotective activity of Nrf2; Sp Aim#2: Test the hypotheses that UbcM2 regulates Keap1 nuclear import and the affinity of Nrf2 for its cognate response elements in the promoters of anti-oxidant genes. Keap1 is the substrate adaptor that targets Nrf2 for degradation; and Sp Aim#3: Test the hypothesis that UbcM2 regulates substrate adaptor exchange on a class of UPS enzymes called cullin-RING E3 ligases (CRLs). In addition, identify the molecular determinants governing polyUb chain synthesis by UbcM2. The proposed experiments utilize a complementary set of cell culture approaches (e.g., siRNA, microinjection/live cell video microscopy, transcription assays), biochemical and biophysical methods (e.g., recombinant pulldowns, in vitro ubiquitylation assays, mass spectrometry, NMR spectroscopy), and in vivo mouse models of oxidative stress. Together, these studies will: (i) define the molecular mechanisms by which UbcM2 modulates Nrf2 stability and activation, (ii) explore the novel idea that Ub E2s can function as redox sensors to mediate stress response pathways, and (iii) bridge gaps in our understanding of how CRLs exchange substrate adaptors. This information will impact both the UPS and oxidative stress fields by identifying new relationships between E2 function and cellular anti-oxidant defenses.